Gap junctions serve an essential role in the passage of molecules from the cytoplasm of one cell to its neighbor in both functional and homeostatic capabilities. They are defined as clusters of closely packed intercellular membrane channels embedded in the plasma membranes of two adjoining cells. The channels are composed of two hexamers of a protein (connexon) from a family of integral membrane proteins known as connexins. Here, we focus on the structure and function of connexin26 (Cx26), the smallest of the family. Mutations in the DNA sequence can result in hereditary sensorineural deafness and account for between one third to one half of the cases of prelingual inherited deafness in Caucasian populations. We have isolated preparations of Cx26 gap junctions in pure and sufficient amounts for biochemical and structural studies. These 2D crystals are amenable to electron microscopy (EM) structure determination and conformational dynamics as revealed with atomic force microscopy (AFM) done under hydrated conditions. In SPECIFIC AIM 1, we will determine the structure of the Cx26 hemichannel beyond 10 Angstroms using state of the art cryo-EM and improvements on image processing procedures. This involves improving specimen preparation, imaging at either liquid nitrogen or liquid helium temperature and implementation of a combined single particle/ 2D crystallographic approach to circumvent imperfect crystal lattices. In SPECIFIC AIM 2, we will construct Cx26 wild type and mutant cell lines with a tetracysteine domain genetic tag to improve isolation with FlAsH ligand affinity bead purification, stably express these in HeLa cells or in baculovirus-infected Sf9 insect cells and isolate the gap junctions or connexons for structural analysis using the methods developed in Specific Aim 1. We will construct two Cx26 mutants (P97L and T135A), each containing a single point mutation in one of the transmembrane helices that changes the effective pore properties. These mutations should be reflected in conformational changes in the 3D structure. In SPECIFIC AIM 3, we will expand coordinated AFM/EM experiments for visualizing conformational changes due to treatments known to close or alter gap junction mediated communication. Preliminary AFM images have visualized conformational changes at submolecular resolution. We have chosen five treatments that known to induce closure of Cx26 channels or hemichannels and are physiologically relevant. Conformational changes identified by AFM imaging will be further imaged using EM. Each of these goals is intended to complement the others and lead to structural and physiological models of Cx26 germane to the entire connexin family.